Description of a Swine Infant Model of Volume-Controlled Hemorrhagic Shock

The experiments in this protocol were approved by the Institutional Ethics Committee for Animal Research of the Gregorio Marañón University Hospital, Madrid, Spain, and the Agriculture and the Environment Council of the Madrid Autonomous Government (permit number: 12/0013). European and Spanish guidelines for ethical care and the use of experimental animals were applied throughout the study. The experiments were performed in the Department of Experimental Medicine and Surgery, Gregorio Marañón University Hospital, Madrid, Spain.

NOTE: The animal model chosen consisted of healthy 2-3-month-old (8-12 kg) minipigs (Sus scrofa domestica). Minipigs are the result of a crossing of three different breeds that make them suitable for biomedical research. The animals are near-identical lines and are provided by a specifically authorized breeding facility in Madrid (IMIDRA), which keeps the maintenance of three homozygous genetic lines in purity. Male and female animals were used interchangeably. Animals were fed a standard swine diet and observed for a minimum of 2 days to ensure good health. Food, but not water, was withdrawn the night before the procedures to reduce the risk of aspiration. A typical experiment requires approximately 6 h to complete, including 30 min for anesthesia induction and surgical preparation, 60 min for instrumentation, 30 min for recovery, 60 min for hemorrhage induction and posterior stabilization, 30 min for resuscitation, and 120 min for follow-up.

1. Anesthesia, intubation, and mechanical ventilation

1. Premedicate the pig with an intramuscular injection of ketamine (10 mg/kg) and atropine (0.02 mg/kg) in the lateral region of the neck, behind the ear, or in the posterior femoral region.
   NOTE: Anticholinergic drugs, such as atropine, are useful as swine may salivate excessively under anesthesia¹⁰. In our experience, this dose of ketamine is enough to reduce stress and induces adequate sedation and analgesia in pigs without adverse effects. However, if the animal is not properly sedated or if the distance from the housing to the operating room is long, another dose of ketamine (10 mg/kg) can be administered safely.
   CAUTION: Gloves are necessary when handling animals.
2. Transport the sedated animal to the operating room and place it on a surgical table provided with a warming blanket.
3. Measure peripheral oxygen saturation (Sp02) with a sensor clipped to the pig's ear and initiate continuous three-lead electrocardiographic (EKG) monitoring.
4. Disinfect the skin with at least 3 alternating rounds of povidone-iodine or chlorhexidine scrub and alcohol. Insert a peripheral vein catheter (22-24 G) into the ear vein. Disinfect the skin priorly with an antiseptic solution.
5. Induce anesthesia by an intravenous injection of fentanyl (5 µg/kg), propofol (4 mg/kg), and atracurium (0.5 mg/kg). Once spontaneous breathing disappears and the absence of reflexes is confirmed, place the animal in the dorsal recumbency position and immediately initiate hand-bag mask ventilation with a dog mask with fraction of inspired oxygen (Fi02) set at 100%.
   NOTE: In order to reduce the risk of accidental awareness related to the use of neuromuscular blockers, anesthetic agents with known effectivity in swine and with doses on the higher limit should be used to ensure an adequate level of anesthesia. Additionally, continuously monitor cardiovascular signs such as heart rate, blood pressure, and body temperature, and administer neuromuscular blockers only when withdrawal reflexes are absent (pedal withdrawal, palpebral reflexes, and jaw tone) and muscle tone is relaxed.
6. Perform endotracheal intubation. At least two operators are needed for this procedure.
   1. Ensure that the basic equipment and surgical tools needed for endotracheal intubation are ready: tie gauze to open the mouth and secure the tube, veterinary laryngoscope with a straight blade between 17 and 25 cm long, a common endotracheal tube (ID 4-5), stylet, syringe with air, and adhesive tape.
   2. Pull out the tongue slightly and hold the jaw open using tie gauze placed behind the upper and lower canine teeth.
   3. Perform a laryngoscopy and, once the epiglottis is visible, use the tip of the laryngoscope to press the epiglottis upward toward the base of the tongue.
      NOTE: If the epiglottis is stuck to the soft palate, it can be displaced dorsally with the tip of the tube. Operator 1 performs step 1.6.2 while operator 2 performs step 1.6.3.
   4. Once the vocal cords are visualized, gently advance the tube with slight rotation into the trachea.
      NOTE: The narrowest point of the trachea is on the subglottic level. If tube insertion is difficult, try slight rotation or a smaller tube.
   5. Remove the stylet and use a 5 mL syringe to inflate the cuff.
   6. Ensure placement of the endotracheal tube by observing symmetrical chest rise, adequate oxygen saturation (95%-100%), and a proper waveform and end-tidal CO₂ (EtCO2) reading.
      CAUTION: Pigs are very susceptible to laryngospasm and edema of the larynx mucosa, and laryngeal perforation may even occur after several attempts of intubation or if sedation is inadequate¹⁰.
7. After confirming intubation, initiate mechanical ventilation using a mechanical ventilator with a respiratory rate of 20 breaths per minute, tidal volume of 8 mL/kg, FiO2 of 40%, and positive end-expiratory pressure of 4 cm H2O. Adjust ventilation to achieve a partial pressure of carbon dioxide (PaCO2) between 35 and 45 mmHg.
8. Maintain deep anesthesia throughout the experiment via a continuous infusion of fentanyl (10 µg/kg/h), propofol (10 mg/kg/h), and atracurium (2 mg/kg/h).

2. Instrumentation

1. Prepare the femoral area for vessel catheterization. Use bandages to pull the legs back and disinfect the inguinal area with at least 3 alternating rounds of povidone-iodine or chlorhexidine scrub and alcohol.
2. Assess the femoral vessels with an ultrasound and use the Doppler technique to distinguish between the artery and the vein. Depending on the size of the vein, insert a 5.5-7.5 French (F) central venous catheter with three ports in one of the femoral veins under continuous ultrasound view and using the Seldinger technique^11,12.
3. Immediately after central venous catheter placement, connect a transducer system to measure the central venous pressure.
4. Ensure that an electrolyte with glucose infusion (20 mL/h) is connected to one of the central line ports and that a maintenance saline infusion (5 mL/h) is infused via the remaining port to prevent occlusion of the catheter.
5. Use the same technique to cannulate the opposite femoral artery with a 4 F arterial catheter specifically designed for cardiac output monitoring. Perform a blood gas test to establish the correct position of the catheter if ultrasound confirmation is not possible.
   NOTE: In case of significant spasm or hematoma, cross over to the contralateral femoral artery.
6. Once the arterial catheter is inserted, connect the arterial wire of the cardiac output monitor system and the arterial transducer directly to the monitor port. Simultaneously connect the venous measuring unit of the monitor to the central venous transducer.
   NOTE: The cardiac output monitor used in this experiment is specified in the Table of Materials. For setup, calibration, and measures, see the manufacturer instructions¹³.
7. Ensure both the venous and arterial transducers are calibrated to zero.
8. Expose the left internal carotid artery and left external jugular vein via the cut-down technique.
   1. Ensure the necessary equipment and surgical tools are available: scalpel, blunt-tip surgical scissors, tissue forceps, small self-retaining tissue retractor, needle holder, surgical swaps, suture with needle, one 18 G IV cannula, one 5 F catheter sheath with an introducer, and one Seldinger guide wire.
   2. With the animal in the dorsal recumbency position, disinfect the neck skin with an antiseptic solution.
   3. Use a scalpel to make an ~10 cm left paratracheal incision, bisecting a line between the manubrium and the angle of the jaw.
   4. To expose the external jugular vein, dissect the tissue lateral to the SCM and isolate the vein from the surrounding fascia.
   5. After being isolated, use two non-absorbable silk sutures (USP-0) looped around the vein to fixate the vessel prior to the puncture.
   6. Incise the vein with a Venflon needle (18 G). Once inside the vein, retract the needle and insert the guide wire through the Venflon tube.
   7. Remove the Venflon tube and insert the sheath with the introducer (5 F) over the wire. After insertion, remove both the introducer and the wire.
   8. Immediately after insertion, rinse the sheaths with 0.9% NaCl (5 mL/h) to prevent thrombus formation.
   9. Tie up the proximal silk suture around the sheath to fix it. After that, secure the handle of the sheath to the SCM and close the skin with staples.
9. Following surgical preparation, allow the animals to stabilize for 30 min before obtaining the baseline monitoring values and blood samples.
10. Maintain the blood temperature at 37-39 ˚C using a thermal blanket and an overhead warmer throughout the experiment.
    ​NOTE: Temperature is measured with a thermistor located at the thermodilution arterial catheter tip.

3. Hemodynamic and perfusion monitoring

1. Monitor the EKG, peripheral oxygen saturation, respiratory volumes and pressures, and Fi02.
2. Connect a spirometer between the endotracheal tube and a multiparameter monitor to measure qualitative and quantitative EtC02.
   NOTE: For more information about the multiparameter monitor, see the Table of Materials.
3. Use near-infrared spectroscopy (NIRS) to monitor the brain tissue oxygenation index (bTOI) and splanchnic tissue oxygenation index (aTOI). Place the sensors on the skin of the forehead and the anterior abdominal wall (subhepatic region).
   NOTE: Do not place the brain sensor in the midline, as it may be contaminated with the superior sagittal sinus venous blood¹⁴.
4. Connect the blood flow probe attached to the internal carotid artery to a flow monitor to measure the carotid blood flow (CaBF).
5. Place a laser Doppler sensor over the skin of the anterior abdominal wall for continuous measurement of the cutaneous tissular blood flow (CuTBF).
   NOTE: For more information about the carotid and cutaneous tissular blow flow sensors, see the Table of Materials.
6. Record the following parameters at baseline and every 30 min: blood temperature, inspiratory tidal volume, EtCO2, cardiac rhythm, heart rate (HR), systolic and diastolic blood pressures, mean arterial blood pressure (MAP), shock index (HR/systolic blood pressure)¹⁵, central venous pressure, cardiac index (CI), global end diastolic volume index (GEDVI), stroke volume index (SVI), left ventricular contractility (Dt/Dpmax), systemic vascular resistance index (SVRI), extravascular lung water index (ELWI), pressure pulse variation (PPV), peripheral hemoglobin saturation, central venous saturation (ScvO2), cerebral (bTOI) and splanchnic (aTOI) tissue oxygenation index by NIRS, CaBF, and CuTBF.
7. To obtain CI values, infuse 5 mL boluses of 0.9% normal saline at a temperature below 8 °C through the central venous catheter. Record the average of two consecutive measures.
8. Determine the arterial and venous blood gas profiles and lactate concentration every 30 min by drawing 0.3 mL of blood from the femoral vessels. Perform standard complete blood counts, coagulation studies, and biochemistry at baseline, after hemorrhage induction, and at the end of the experiment.
9. After each blood draw, flush the lines with 0.5 mL of 100 IU/mL heparin.

4. Hemorrhagic shock induction

1. Once a steady state is achieved after instrumentation and baseline data has been gathered, induce hypovolemic shock by withdrawing 30 mL/kg of blood from the jugular vein over 30 min.
2. Allow a 30 min period for stabilization. Do not make resuscitation efforts during this period to emulate the delay in arrival of emergency medical teams.

5. Infusion and follow-up

1. After the stabilization period, infuse a bolus of a volume expander or a vasoactive agent over a 30 min period.
   NOTE: Examples of volume expanders and vasoactive agents tested are normal saline, hypertonic albumin, angiotensin, and terlipressin. In this study, 30 mL/kg normal saline (NS) (n = 13), 15 mL/kg 5% albumin plus 3% hypertonic saline (AHS) (n = 13), or a single intravenous bolus of 15 µg/kg terlipressin plus 15 mL/kg 5% albumin plus 3% hypertonic saline (TAHS) (n = 13) were used.
2. After the infusion, follow up the animal for 120 min. Record the hemodynamic parameters and obtain blood samples every 30 min for arterial and venous blood gas profiles and lactate concentration determination. Do not make resuscitation efforts during this period.

6. End of the experiment and euthanasia

1. Once the experiment is completed, use a sedative overdose (5 µg/kg fentanyl and 10 mg/kg propofol) and an intravenous injection of potassium chloride (2 mEq/kg) to sacrifice all successfully resuscitated animals.
2. Confirm the absence of circulation by asystole or pulseless electrical activity on a continuous EKG display, the absence of pulsatile flow during invasive arterial pressure monitoring, and the absence of other vital signs.
3. If, during the follow-up period, arterial blood pressure decreases below 25 mmHg, sacrifice the animal to avoid further suffering.